PROJECT SUMMARY Cytosolic lipid droplets are unique among cellular organelles. These essential structures, found in nearly all eukaryotic cells, serve several functions besides fat storage, including serving as a depot for hormones and signaling molecules, providing phospholipid precursors for other organelles, innate immunity, and as a platform for assembly of pathogens. Unlike other organelles, droplets contain a single phospholipid leaflet which surrounds their hydrophobic cores. Despite the fact that they are the basis of both obesity, which affects two thirds of Americans, and lipodystrophy, an often fatal disease caused by the inability to properly store fat, there is still sketchy knowledge of their assembly. Recent progress has shown that, while droplets can form spontaneously, a few proteins and lipids are involved in regulating this process and ensuring that fully functional droplets are produced. While these components have been identified, much is unknown about their functions. This proposal seeks to test hypotheses regarding the mechanism of action of Pet10p (a yeast perilipin), seipin, and Scs3p (a Fit2 protein), and to gain molecular insight into how they function together. A driving hypothesis is that Pet10p collaborates in trans (i.e., at the bud rather than at the ER/droplet junction) with seipin, Scs3p, and diacylglycerol to promote and regulate droplet assembly. In Aim 1, focusing on Pet10p, the multiple phenotypes in PET10-deletion cells with be matched with specific domains to probe function. The basis of the stabilizing effect of Pet10p on droplets will be elucidated, and whether the same activity is responsible for its role in droplet formation will be determined. The focus of Aim 2 will be a deeper understanding the function of seipin, including mapping function to structure, control of targeting proteins to droplets, and its role in vectorial budding of droplets. Studies to elucidate structure from cryo-EM images will also be initiated. Aim 3 will focus on the collaboration of seipin, Pet10p and Scs3p on early steps of droplet budding. Whether seipin attracts Pet10p through seipin?s amino terminal will be determined, and the role of Pet10p and Scs3p on budding will be studied. The order of arrival at the nascent structure will be probed. Overall, the proposed research will elucidate important functions of the droplet assembly factors, and, for the first time, study how they interact.